This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A) The bacterial 30S ribosome is composed of a 1542 nucleotide 16S ribosomal RNA and 20 small ribosomal proteins. It is responsible for mRNA decoding in association with the 50S subunit during protein synthesis. The 30S subunit from E. coli can be reconsituted in vitro from purified components. An assembly map has been developed that contains both parallel protein binding and sequential protein binding as mechanistic features. The emerging picture of 30S assembly is that it is a statistical distribution of assembly pathways, but that there are favored routes over the assembly landscape. Intermediates with subsets of proteins bound accumulate during the assembly, but these intermediates eventually converge on the final completed subunit. We are interested in measuring the time dependence of the populations of intermediates by visualizing large number of ribosomal particles during in vitro reconstitution. B) The 30S subunit is a paradigm for spontaneous formation of a molecular assembly. It is one of the best characterized large systems both structurally and from the standpoint of assembly. Understanding the detailed mechanism of 30S assembly will provide insights into the assembly of many classes of molecular machines involving large numbers of components.